The present invention relates to an improved integrin-targeting vector that has enhanced transfection activity.
Gene therapy and gene vaccination are techniques that offer interesting possibilities for the treatment and/or prophylaxis of a variety of conditions, as does anti-sense therapy. Such techniques require the introduction of a DNA of interest into target cells. The ability to transfer sufficient DNA to specific target cells remains one of the main limitations to the development of gene therapy, anti-sense therapy and gene vaccination. Both viral and non-viral DNA delivery systems have been proposed. In some cases RNA is used instead of DNA. Receptor-mediated gene delivery is a non-viral method of gene transfer that exploits the physiological cellular process, receptor-mediated endocytosis to internalise DNA. Receptor-mediated non-viral vectors have several advantages over viral vectors. In particular, they lack pathogenicity; they allow targeted gene delivery to specific cell types and they are not restricted in the size of nucleic acid molecules that can be packaged. Gene expression is achieved only if the nucleic acid component of the complex is released intact from the endosome to the cytoplasm and then crosses the nuclear membrane to access the nuclear transcription machinery. However, transfection efficiency is generally poor relative to viral vectors owing to endosomal degradation of the nucleic acid component, failure of the nucleic acid to enter the nucleus and the exclusion of aggregates larger than about 150 nm from clathrin coated vesicles.
Integrins are a super-family of heterodimeric membrane proteins consisting of several different xcex1 and xcex2 subunits. They are important for attachment of cells to the extracellular matrix; cell-cell interactions and signal transduction. Integrin-mediated cell entry is exploited for cell attachment and entry by a number of intracellular pathogens including Typanosoma cruzi (Fernandez et al., 1993), adenovirus (Wickham et al., 1993), echovirus (Bergelson et al., 1992) and foot-and-mouth disease virus (Logan et al., 1993) as well as the enteropathogen Y. pseudotuberculosis (Isberg, 1991). Egg-sperm fusion is also integrin mediated. Intensive study of the invasin-integrin mediated internalisation process of Yersinia pseudotuberculosis demonstrated that, for efficient cell entry, integrin-binding ligands should have a high binding affinity and a non-polar distribution (Isberg, 1991). Integrin-mediated internalisation proceeds by a phagocytic-like process allowing the internalisation of bacterial cells one to two micrometers in diameter (Isberg, 1991). Targeting of non-viral vectors to integrins, therefore, has the potential to transfect cells in a process that mimics infection of cells by pathogens and avoids the size limitation imposed by clathrin-coated vesicles in receptor-mediated endocytosis.
A further advantage of integrin-mediated vectors is that a large number of peptide ligands for integrin receptors have been described, including sequences derived from natural protein ligands [Verfaille, 1994 #635; Wang, 1995 #645; Staatz, 1991 #539; Pierschbacher, 1984 #314; Massia, 1992 #86, Clements et al. 1994 and Lu et al, 1993] or selected from phage display libraries (Koivunen et al. 1995; 1993; 1994; O""Neil et al. 1992; Healy et al 1995; Pasqualani et al. 1995).
The conserved amino acid sequence arginine-glycine-aspartic acid (RGD) is an evolutionarily conserved feature of many, but not all, natural integrin-binding ligands such as extracellular matrix proteins and viral capsids. Peptides, particularly those containing cyclic-RGD domains can also bind integrins. Peptides containing cyclic-RGD domains are particularly suitable ligands for vectors since they bind to integrins with higher affinities than linear peptides (Koivunen et al. 1995). Hart et al. have demonstrated previously that multiple copies of a cyclic RGD peptide displayed in the major coat protein subunit of fd filamentous phage particles, approximately 900 nm in length, are internalised efficiently by cells in tissue culture in an integrin-mediated manner (Hart et al., 1994). The phage particles were probably internalised by a phagocytic-like process as their size would exclude them from endocytosed vesicles (Hart et al., 1994).
The cyclic RGD-containing peptide GGCRGDMFGCGG[K]16 [SEQ.ID.NO.:1] was synthesised with a sixteen-lysine tail for complex formation with plasmid DNA (Hart et al., 1995). Significant levels of integrin-mediated gene expression were achieved in epithelial cell lines with the vector GGCRGDMFGCG[K]16 [SEQ.ID.NO.:2] (Hart et al., 1995) and the vectors GGCRGDMFGC[K]16 [SEQ.ID.NO.:3] (WO96/15811). A similar peptide [K]16GACRGDMFGCA [SEQ.ID.NO. :4], which has the sixteen-lysine domain at the N-terminus and which is easier to synthesise than the prototype peptide (WO96/15811 and Hart et al., 1997) generated better transfection levels. Integrin mediated gene expression was generally achieved at levels of about 1 to 10%. The presence of chloroquine in the transfection medium gave some enhancement of transfection in some but not all cell lines tested.
The present invention is based on the surprising observation that inclusion of a lipid component in the oligolysine/-peptide/DNA complex increases levels of transfection of DNA from about 1 to 10% to about 50 to almost 100%. Not only is the level of transfection increased dramatically but, contrary to previous experience, the increase is observed in all cell lines tested, including endothelial, epithelial and tumour cell lines.
The present invention provides a complex that comprises
(i) a nucleic acid, especially a nucleic acid encoding a sequence of interest,
(ii) an integrin-binding component,
(iii) a polycationic nucleic acid-binding component, and
(iv) a lipid component.
The complex is a transfection vector.
The nucleic acid may be obtained from natural sources, or may be produced recombinantly or by chemical synthesis. It may be modified, for example, to comprise a molecule having a specific function, for example, a nuclear targeting molecule. The nucleic acid may be DNA or RNA. DNA may be single stranded or double stranded. The nucleic acid may be suitable for use in gene therapy, in gene vaccination or in anti-sense therapy. The nucleic acid may be or may relate to a gene that is the target for particular gene therapy or may be a molecule that can function as a gene vaccine or as an anti-sense therapeutic agent. The nucleic acid may be or correspond to a complete coding sequence or may be part of a coding sequence.
Alternatively, the nucleic acid may encode a protein that is commercially useful, for example industrially or scientifically useful, for example an enzyme; pharmaceutically useful, for example, a protein that can be used therapeutically or prophylactically as a medicament or vaccine; or diagnostically useful, for example, an antigen for use in an ELISA. Host cells capable of producing commercially useful proteins are sometimes called xe2x80x9ccell factoriesxe2x80x9d.
Appropriate transcriptional and translational control elements are generally provided. For gene therapy, the nucleic acid component is generally presented in the form of a nucleic acid insert in a plasmid or vector. In some cases, however, it is not necessary to incorporate the nucleic acid component in a vector in order to achieve expression. For example, gene vaccination and anti-sense therapy can be achieved using a naked nucleic acid.
The nucleic acid is generally DNA but RNA may be used in some cases, for example, in cancer vaccination. The nucleic acid component is referred to below as the plasmid component or component xe2x80x9cEDxe2x80x9d.
The integrin-binding component is any component that is capable of binding specifically to integrins found on the surface of cells. The integrin-binding component may be a naturally occurring integrin-binding ligand, for example, an extra-cellular matrix protein, a viral capsid protein, the bacterial protein invasin, a snake venom disintegrin protein, or an integrin-binding fragment of any such protein. Such integrin-binding proteins and fragments thereof may be obtained from natural sources or by recombinant techniques, but they are difficult to synthesise and purify in large amounts, they require conjugation directly to DNA or RNA or to polycationic elements for DNA or RNA binding, and are immunogenic in vivo.
It is preferable to use integrin-binding peptides, in particular because of their ease of synthesis, purification and storage, their potential for chemical modification, and their potentially low immunogenicity in vivo. Examples of integrin-binding peptides are given in Verfaille, 1994 #635; Wang, 1995 #645; Staatz, 1991 #539; Pierschbacher, 1984 #314; Massia, 1992 #86, Clements et al. 1994 and Lu et al, 1993; and in Koivunen et al. 1995; 1993; 1994; O""Neil et al. 1992; Healy et al 1995; and Pasqualani et al. 1995.
As indicated above, peptides containing the conserved amino acid sequence arginine-glycine-aspartic acid (RGD) bind with high affinity to integrins. Accordingly, peptides comprising the RGD sequence are particularly useful. The affinity between integrin and peptide ligands is influenced by the amino acid sequence flanking the RGD domain. Peptides having a cyclic region in which the conformational freedom of the RGD sequence is restricted generally have a higher affinity for integrin receptors than do their linear counterparts. Such cyclic peptides are particularly preferred. Cyclic peptides may be formed by the provision of two cysteine residues in the peptide, thus enabling the formation of a disulphide bond. A cysteine residue may be separated from the RGD sequence by one or more residues, for example, up to six residues, or may be immediately adjacent to the RGD sequence, although preferably both cysteines are not immediately adjacent to the ends of the RGD sequence.
An example of an amino acid sequence that will permit cyclisation by disulphide bond formation is CRGDMFGC [SEQ.ID.NO.:5]. A peptide that consists of or comprises the sequence CRGDMFGC may advantageously be used as an integrin-binding peptide according to the present invention. Examples of peptides that comprises the sequence CRGDMFGC and that are effective integrin-binding ligands are the peptides GGCRGDMFGC [SEQ.ID.NO.:6], GGCRGDMFGCG [SEQ.ID.NO.:7], GGCRGDMFGCA [SEQ.ID.NO.:8] and GACRGDMFGCA [SEQ.ID.NO.:9].
The peptide GACDCRGDCFCA [SEQ.ID.NO.:10] has the potential to form two disulphide bonds for stabilising the RGD loop. That peptide and others having the potential to form two RGD-stabilising disulphide bonds, may be particularly useful as integrin-binding ligands according to the present invention.
However, not all integrin-binding peptides contain the conserved RGD sequence. For example, the peptides GACRRETAWACA [SEQ.ID.NO.:11] and GACRRETAWACG [SEQ.ID.NO.:12] are integrin-specific peptides. Other peptides comprising the sequence CRRETTAWAC [SEQ.ID.NO.:13] may be used, as may other non-RGD peptides, particularly those that have the potential for disulphide bond formation.
Peptide sequences may be designed on the basis of known ligands, for example, on the basis of integrin-binding domains of naturally-occurring integrin-binding ligands, or on the basis of known peptides that bind to integrins.
As stated above integrins are a family of heterodimeric proteins found on the surface of cells. They consist of several different xcex1 and xcex2 subunits. Some integrins are found on may types of cells, others are more specific, for example, xcex15 and xcex1v integrins are widespread and are found on a diverse range of cells. Integrin-binding ligands can vary in their affinity for different integrins. For example, GACRGDMFGCA [SEQ.ID.NO.:9] (peptide 1) has affinity for xcex15 and xcex1v integrins but is non-specific (O""Neil et al. 1992, Hart et al. 1997). GACDCRGDCFCA [SEQ.ID.NO.:10] (peptide 5) has high affinity for integrin xcex1v but is not xcex1v-specific (Koivunen et al. 1995; Hart et al. 1997). GACRRETAWACG [SEQ.ID.NO.:12] (peptide 6) however, which does not contain the conserved RGD region, is xcex15xcex21-specific (Koivunen et al. 1995). Various integrin-binding peptides and their integrin specificity are set out in the Table below:
It should be noted that the use of a lipid component according to the present invention greatly enhances transfection for all peptides and all cell types tested, unlike other enhancement techniques that have been tried, for example, chloroquine, which enhance transfection to a small extent in some but not all cell types tested.
The polycationic nucleic acid-binding component is any polycation that is capable of binding to DNA or RNA. The polycation may have any number of cationic monomers provided the ability to bind to DNA or RNA is retained. For example, from 3 to 100 cationic monomers may be present, for example, from 10 to 20, especially about 16. An oligolysine is particularly preferred, for example, having from 10 to 20 lysine residues, for example, from 15 to 17 residues, especially 16 residues i.e. [K]16.
The polycationic DNA or RNA-binding component may advantageously be linked or otherwise attached to the integrin-binding component. A combined integrin-binding component/polycationic DNA or RNA-binding component may be referred to below as component xe2x80x9cIxe2x80x9d. For example, a polycationic DNA or RNA-binding component may be chemically bonded to an integrin-binding component, for example, by a peptide bond in the case of an oligolysine. The polycationic component may be linked at any position of the integrin-binding component. Preferred combinations of integrin-binding component and polycationic DNA or RNA-binding component are an oligolysine, especially [K]16, linked via a peptide bond to a peptide, for example, a peptide as described above.
The lipid component may be or or may form a cationic liposome. The lipid component may be or may comprise one or more lipids selected from cationic lipids and lipids having membranae destabilising or fusogenic properties, especially a combination of a cationic lipid and a lipid that has membrane destabilising properties.
A preferred lipid component (xe2x80x9cLxe2x80x9d) is or comprises the neutral lipid dioleyl phosphatidylethanolamine, referred to herein as xe2x80x9cDOPExe2x80x9d. DOPE has membrane destabilising properties sometimes referred to as xe2x80x9cfusogenicxe2x80x9d properties (Farhood et al. 1995). Other lipids, for example, neutral lipids, having membrane destabilising properties, especially membrane destabilising properties like those of DOPE may be used instead of or as well as DOPE.
Other phospholipids having at least one long chain alkyl group, for example, di(long alkyl chain)phospholipids may be used. The phospholipid may comprise a phosphatidyl group, for example, a phosphatidylalkanolamine group, for example, a phosphatidylethanolamine group.
A further preferred lipid component is or comprises the cationic lipid N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride, referred to herein as xe2x80x9cDOTMAxe2x80x9d. DOTMA has cationic properties. Other cationic lipids may be used in addition to or as an alternative to DOTMA, in particular cationic lipids having similar properties to those of DOTMA. Such lipids are, for example, quaternary ammonium salts substituted by three short chain alkyl groups, and one long chain alkyl group. The short chain alkyl groups may be the same or different, and may be selected from methyl and ethyl groups. At least one and up to three of the short chain alkyl group may be a methyl group. The long alkyl chain group may have a straight or branched chain, for example, a di(long chain alkyl)alkyl group.
Another preferred lipid component is or comprises the lipid 2,3-dioleyloxy-N-[2-(spermidinecarboxamido)ethyl]-N,N-dimethyl-1-propanaminiumtrifluoridoacetate, referred to herein as xe2x80x9cDOSPAxe2x80x9d. Analogous lipids may be used in addition to or as an alternative to DOSPA, in particular lipids having similar properties to those of DOSPA. Such lipids have, for example, different short chain alkyl groups from those in DOSPA.
A preferred lipid component comprises DOPE and one or more other lipid components, for example, as described above. Especially preferred is a lipid/component that comprises a mixture of DOPE and DOTMA. Such mixtures form cationic liposomes. An equimolar mixture of DOPE and DOTMA is found to be particularly effective. Such a mixture is known generically as xe2x80x9clipofectinxe2x80x9d and is available commercially under the name xe2x80x9cLipofectinxe2x80x9d. The term xe2x80x9clipofectinxe2x80x9d is used herein generically to denote an equimolar mixture of DOPE and DOTMA. Other mixtures of lipids that are cationic liposomes having similar properties to lipofectin may be used. Lipofectin is particularly useful as it is effective in all cell types tested.
A further preferred lipid component comprises a mixture of DOPE and DOSPA. Such mixtures also form cationic liposomes. A mixture of DOPE and DOSPA in a ratio by weight 3:1 DOSPA:DOPE is particularly effective. Such a mixture, in membrane filtered water, is available commercially under the name xe2x80x9cLipofectaminexe2x80x9d. Mixtures comprising DOPE, DOTMA and DOSPA may be used, for example, mixtures of lipofectin and lipofectamine.
Other cationic lipids are available commercially, for example, DOTAP (Boehringer-Mannheim) and lipids in the Tfx range (Promega). DOTAP is N-[1-(2,3-diolyloxy)propyl]-N,N,N-tri-methylammonium methylsulphate. The Tfx reagents are mixtures of a synthetic cationic lipid [N,N,Nxe2x80x2,Nxe2x80x2-tetramethyl-N,Nxe2x80x2-bis(2-hydroxyethyl)-2,3-di(oleoyloxy)-1,4-butanediammonium iodide and DOPE. All the reagents contain the same amount of the cationic lipid component but contain different molar amounts of the fusogneic lipid, DOPE.
However, lipofectin and lipofectamine appear to be markedly more effective as the lipid component in LID complexes of the present invention than are DOTPA and Tfx agents.
The effectiveness of a putative integrin-binding component, polycationic DNA or RNA-binding component, or of lipid component may be determined readily using the methods described herein.
The efficiency of transfection using a complex of the invention is influenced by the ratio lipid component:integrin-binding component:DNA or RNA. For any chosen combination of components for any particular type of cell to be transfected, the optimal ratios can be determined simply by admixing the components in different ratios and measuring the transfection rate for that cell type, for example, as described herein.
For example, a combination consisting of a pGL2 plasmid, which is a plasmid encoding luciferase (a reporter gene) under an SV40 promoter as DNA component (D), [K]16GACRGDMFGCA [SEQ.ID.NO.:17] ([K]16-peptide 1) as a combined integrin-binding component/polycationic DNA binding component (I), and lipofectin (DOPE:DOTMA 1:1 molar ratio) as the lipid component (L) was tested to find the optimal ratio of components. Complexes formed with 1 xcexcg of lipofectin (L) and 4 xcexcg of [K]16-peptide (I) per 1 xcexcg of plasmid (D) were 100-fold more active than complexes lacking lipofectin. Addition of larger amounts of lipofectin reduced transfection activity in a lipofectin dose-dependent manner.
An optimal transfection ratio of 0.75 xcexcg of lipofectin (L) per 4 xcexcg of the (K]16-peptide integrin-binding component/-poly-cationic DNA or RNA-binding component (I) per 1 xcexcg plasmid DNA or RNA (nucleic acid component, D) was found for three different cell lines namely melanoma cell, endothelial cells and epithelial cells. That ratio was subsequently found to be effective for other different cell lines and for other oligolysine-peptides. A ratio L:I:D of 0.75:4:1 by weight corresponds to a molar ratio of 0.5 nmol lipofectin: 1.25 nmol [K]16 -peptide 6: 0.25 pmol plasmid pGL2-control. A ratio L:I:D of 0.75:4:1 by weight, or the corresponding molar ratio are preferred when lipofectin is used as the lipid component.
For a combination of components in which lipofectin is replaced by lipofectamine (DOPE/DOSPA), the optimal ratio was found to be 12 xcexcg lipofectamine: 4 xcexcg [K]16-peptide 6: 1 xcexcg plasmid DNA or RNA. A ratio of L:I:D of 12:4:1 by weight, or the corresponding molar ratio, is appropriate for lipofectamine-containing complexes. Optimal ratios for other systems may be determined analogously.
Lipofectin and lipofectamine appear to be particularly effective in enhancing transfection. Lipofectin has the advantage that only very small amounts are required. Any side effects that may occur are therefore minimised. As indicated above, the optimal weight ratio of components L:I:D when using lipofectamine is 12:4:1. With lipofectin the optimal ratio is only 0.75:4:1.
The present invention provides a process for the production of a transfection complex of the present invention, which comprises admixing components (i), (ii), (iii) and (iv).
Although the components may be admixed in any order, it is generally preferable that the lipid component is not added last. In the case where there is a combined integrin-binding component/polycationic DNA or RNA-binding component it is generally preferable to combine the components in the following order: lipid component; combined integrin-binding/polycationic DNA or RNA-binding component; DNA or RNA component, for example, in the order: lipofectin, oligolysine-peptide component, DNA or RNA component.
The present invention also provides a mixture comprising an integrin-binding component, a polycationic nucleic acid-binding component, and a lipid component.
Such a mixture may be used to produce a nucleic acid-containing transfection complex of the invention by the incorporation of a nucleic acid with the mixture, for example, by admixture. Alternatively, the mixture of the invention may be used for the production of a complex which comprises, instead of the nucleic acid component, any other component that is capable of binding to the polycationic nucleic-acid binding component, for example, a protein.
The present invention further provides a process for the production of a complex of the present invention, which comprises admixing a nucleic acid with a mixture of the invention.
The individual components of a mixture of the invention are each as described above in relation to the complex of the invention. The preferred components, preferred combinations of components, preferred ratios of components and preferred order of mixing, both with regard to the mixture and to the production of a complex, are as described above in relation to the complex of the invention.
A mixture of the present invention preferably comprises an equimolar mixture of DOPE and DOTMA (lipofectin) as the lipid component and an oligolysine-peptide especially a [K]16-peptide as a combined integrin-binding/nucleic acid-binding component. The preferred molar ratio lipofectine:oligolysine-peptide is 0.75:4.
The present invention provides a method of transfecting a cell with a nucleic acid, which comprises contacting the cell in vitro or in vivo with a complex of the present invention.
The present invention also provides a process for expressing a nucleic acid in a host cell, which comprises bringing the cell into contact with a complex of the present invention. The host cell is then cultured under conditions that enable the cell to express the nucleic acid.
The present invention further provides a process for the production of a protein, which comprises contacting a host cell in vitro or in vivo with a complex of the present invention, allowing the cell to express the protein, and obtaining the protein. The host cell may be transfected in vitro with a nucleic acid by means of a complex of the present invention and cultured, the protein being obtained either from the host cell or from the culture medium.
The present invention further provides a cell transfected with a complex of the present invention, and also the progeny of such a cell.
The present invention also provides a pharmaceutical composition which comprises a complex of the present invention in admixture or conjunction with a pharmaceutically suitable carrier. The composition may be a vaccine.
The present invention also provides a method for the treatment or prophylaxis of a condition caused in a human or in a non-human animal by a defect and/or a deficiency in a gene, which comprises administering a complex of the present invention to the human or to the non-human animal.
The present invention also provides a method for therapeutic or prophylactic immunisation of a human or of a non-human animal, which comprises administering a complex of the present invention to the human or to the non-human animal.
The present invention also provides a method of anti-sense therapy of a human or of a non-human animal, wherein a complex of the present invention comprising anti-sense DNA is administered to the human or to the non-human animal.
The present invention further provides a complex of the present invention for use as a medicament and/or vaccine, for example for the prophylaxis of a condition caused in a human or in a non-human animal by a defect and/or a deficiency in a gene, for therapeutic or prophylactic immunisation of a human or of a non-human animal, or for anti-sense therapy of a human or of a non-human animal.
The present invention also provides the use of a complex of the present invention for the manufacture of a medicament for the prophylaxis of a condition caused in a human or in a non-human animal by a defect and/or a deficiency in a gene, for therapeutic or prophylactic immunisation of a human or of a non-human animal, or for anti-sense therapy of a human or of a non-human animal.
A non-human animal is, for example, a mammal, bird or fish, and is particularly a commercially reared animal.
The DNA or RNA in the complex of the invention is appropriate for the intended gene therapy, gene vaccination, or anti-sense therapy. The DNA or RNA and hence the complex is administered in an amount effective for the intended purpose.
In a further embodiment, the present invention provides a kit suitable for preparing a mixture of the present invention. Such a kit comprises the following: (i) an integrin-binding component; (ii) a polycationic nucleic acid-binding component, and (iii) a lipid component.
A kit suitable for producing a complex of the present invention may comprise components (i) to (iii) above and (iv) either a nucleic acid or a plasmid or vector suitable for the expression of a nucleic acid, the plasmid or vector being either empty or comprising the nucleic acid.
The components of a kit are, for example, as described above in relation to a complex or a mixture of the present invention. Preferred copmonents are as described above.
A kit generally comprises instructions for the production of a complex or a mixture of the present invention. The instructions preferably indicate the preferred ratios of the components and the preferred order of admixing the components, for example, as described above. A kit may be used for producing a complex suitable for gene therapy, gene vaccination or anti-sense therapy. Alternatively, it may be used for producing a complex suitable for transfecting a host cell with a nucleic acid encoding a commercially useful protein i.e. to produce a so-called xe2x80x9ccell factoryxe2x80x9d.
The kit of the present invention enables the user to produce quickly and easily a highly efficient transfection complex of the present invention using any DNA or RNA of choice.
A kit of the invention may comprises the following components: (a) an integrin-binding component, (b) a polycationic nucleic acid-binding component, (c) a lipid component and (d) a nucleic acid.
Such a kit is suitable for the production of a complex for use, for example, in gene vaccination or anti-sense therapy.
In a kit of the invention the components including the preferred components are, for example, as described above in relation to a complex of the present invention.
The present invention also provides a lipid component as described above for use in increasing the efficiency of transfection of a cell with a nucleic acid, either DNA or RNA, the lipid component being used in combination with an integrin-binding component and a polycationic nucleic acid-binding component.
The present invention also provides the use of a lipid component as described above for the manufacture of a medicament comprising
(i) a nucleic acid, especially a nucleic acid encoding a sequence of interest,
(ii) an integrin-binding component,
(iii) a polycationic nucleic acid-binding component and
(iv) the lipid component.
The medicament may be for gene therapy, gene vaccination, or anti-sense therapy.
The present invention also provides a transfection complex that comprises
(i) a nucleic acid, especially a nucleic acid encoding a sequence of interest,
(ii) an integrin-binding component, and
(iii) a polycationic a nucleic acid-binding component, characterised in that a lipid component, for example as described above, is an additional component of the complex.
The present invention also provides a method for increasing the efficiency of a transfection vector that comprises
(i) a nucleic acid, especially a nucleic acid encoding a sequence of interest,
(ii) an integrin-binding component, and
(iii) a polycationic a nucleic acid-binding component, characterised in that a lipid component, for example as described above, is incorporated as an additional component of the complex.
In each case, the various components are as described above. The lipid component is, for example, a mixture of DOPE and DOSPA or, especially, a mixture of DOPE and DOTMA, in particular an equimolar mixture of DOPE and DOTMA (lipofectin).
Targets for gene therapy are well known and include monogenic disorders, for example, cystic fibrosis, various cancers, and infections, for example, viral infections, for example, with HIV. For example, transfection with the p53 gene offers great potential for cancer treatment. Targets for gene vaccination are also well known, and include vaccination against pathogens for which vaccines derived from natural sources are too dangerous for human use and recombinant vaccines are not always effective, for example, hepatitis B virus, HIV, HCV and herpes simplex virus. Targets for anti-sense therapy are also known. Further targets for gene therapy and anti-sense therapy are being proposed as knowledge of the genetic basis of disease increases, as are further targets for gene vaccination.
Transfection complexes of the present invention have been demonstrated to transfect various different cell types, including endothelial and epithelial cells, and tumour cells. Transfection of all cell types tested including cell types that are particularly reistant to transfection with most plasmid transfection vectors, for example, neuroblastoma cells, primary smooth muscle cells and cardiac myocytes, and haematopoieic cells has been achieved with high efficiency using transfection complexes of the present invention. This enables effective gene therapy, gene vaccination and anti-sense therapy without the previous restrictions as to cell type. For example, transfection with the p53 gene for cancer therapy has great potential but is currently limited by the range of cell types in which effective transfection can be achieved.
The effective tranfection of neuroblastoma cells demonstrates that the complexes of the invention may be used as vaccines or for therapy of neuroblastoma, an important childhood malignancy. The effective transfection of primary smooth muscle cells and cardiac myocytes, which are particularly resistant to plasmid-mediated transfection, demonstrates that diseases and other pathological conditions affecting muscles and the cardiovascular system can now be treated by gene therapy. One such condition is restenosis. After balloon angioplasty plaques reform in 30-50% of cases. A gene that prevents proliferation of cells in blood vessel walls may be introduced using a complex of the present invention to reduce restenosis.
Haematopoietic cells are another cell type that is particularly resistant to plasmid-mediated transfection. The effectiveness of tranfection using a complex of the present invention, which can exceed 60%, now enables gene therapy, gene vaccination and anti-sense therapy of diseases involving haematopoietic cells, including leukaemia and bone marrow stem cell disorders. For example, transfection of a cytokine gene may be used for adjuvant immunotherapy.
Complexes of the invention have been demonstrated to be effective vectors for intracellular transport and delivery of anti-sense oligonucleotides, which enables antiviral and cancer therapy.
Furthermore, complexes of the invention have been demonstrated to be effective for intracellular transport of very large DNA molecules, for example, DNA larger than 125kb, which is particularly difficult using conventional vectors. This enables the introduction of artificial chromosomes into cells.
Transfection at high levels has been demonstrated in vivo, confirming the utility of the complexes of the invention for gene therapy, antisense therapy and gene vaccination. Transfection of the airways, for example, the bronchial epithelium demonstrates utility for gene therapy of, for example, cystic fibrosis and asthma. Transfection of corneal endothelium demonstrates utility for treatment of eye disease affecting the cornea or corneal organ transplants, for example in glaucoma.
The high levels of transfection make the complex of the invention particularly suitable for the production of host cells capable of producing a desired protein, so-called xe2x80x9ccell factoriesxe2x80x9d. For long-term production, it is desirable that the introduced nucleic acid is incorporated in the genome of the host cell, or otherwise stably maintained. That can be readily ascertained. As indicated above, the range of proteins produced in this way is large, including enzymes for scientific and industrial use, proteins for use in therapy and prophylaxis, immunogens for use in vaccines and antigens for use in diagnosis.
The present invention provides a non-viral vector that is capable of high efficiency transfection. In a preferred embodiment, the vector comprises four modular elements; an oligolysine, especially [K]16, DNA or RNA-binding element; a high affinity integrin-binding peptide, for example, a peptide described herein; a DNA or RNA sequence, optionally in a plasmid, and optionally regulated by a viral promoter and an enhancing element; the cationic liposome DOTMA/DOPE (lipofectin). The combination of oligolysine-peptide/DNA or RNA complex with the cationic liposome formulation DOTMA/DOPE is a potent combination. Alternatively a DOPE/DOSPA formulation may be used instead of or in addition to a DOTMA/DOPE formulation. The optimisation of variables associated with complex formation and the mode of transfection by LID complexes has been demonstrated. In addition, analysis by atomic forces microscopy has been carried out to assess the structure of the complexes.
The most important variables in the formation of optimal LID transfection complexes appear to be the ratio of the three components and their order of mixing. The same composition appears to be optimal for all cell lines tested.
The mechanism of action of the complex of the present invention, the reason for the unexpectedly high levels of transfection and the surprisingly wide variety of cells that can be transfected at that high efficiency are not yet understood.
However, the following observations made as a result of the present invention indicate that the role of the lipid component is to enhance the efficiency of transfection mediated by oligolysine-peptide/DNA or RNA complexes:
The level of transfection with LID (lipofectin/[K]16-peptide/plasmid) complexes is three to six fold higher than that with LKD (lipofectin/[K]16/plasmid) complexes prepared with the same charge ratios, or with LD (lipofectin/plasmid) complexes. This indicates that the integrin-targeting moiety, i.e. the peptide, is a significant factor in the transfection efficiency of those complexes.
Optimised LID transfection complexes contain only one seventh of the amount of lipofectin required for optimal transfection with LD complexes. Transfections with low-ratio LD complexes that contain the same ratio of lipofectin to [K]16-peptide/-plasmid as in optimal LID complexes but no [K]16-peptide, did not transfect cells at all. This suggests that the role of lipofectin in LID complexes is to enhance transfection mediated by the integrin receptor-binding peptide.
Furthermore, we have found that both LID and ID complexes both form spherical particles of similar sizes. Optimal LD complexes, however, formed a tubular network with some tubule-associated particles, which suggests a different type of cellular interaction and transfection mechanism from LID and ID transfections.
It is possible that condensation of plasmid DNA or RNA by the oligolysine element of the integrin-targeting oligolysine-peptides and the cationic charge of the complexes may lead to high levels of expression when associated with lipofectin, and the integrin targeting moiety i.e. the peptide is irrelevant. Transfection experiments with LKD complexes, mixed in the same order and the same charge ratios as the LID complexes, were more efficient than LD or KD complexes. To assess the contribution of the relative importance of the oligolysine element and the integrin-targeting peptide domain of the combined integrin-binding component/polycationic DNA or RNA-binding component I, transfection by LID complexes were prepared containing a range of proportions of [K]16 and [K]16integrin targeting peptide 6, [K]16GACRRETAWACG [SEQ.ID.NO.:18]. Transfection expression data indicate higher,efficiencies with complexes in 25 which increasing amounts of [K]16peptide 6 replace [K]16 and a dose-dependency on the amount of integrin-targetting (ligand-binding) domain i.e. peptide 6.
The ratio of components mixed together to form the optimal transfection complex is also informative as to the possible mechanism of lipofectin mediated enhancement. The DOTMA element of lipofectin is cationic, which may enhance the activity of the complex, while DOPE may have the ability to destabilise the endosomal membrane (Farhood et al., 1995) enhancing endosomal release of plasmid DNA or RNA. The components of the LID complexes are mixed together in constant optimal ratios. It is assumed that the particles formed also contain these elements in the same proportions. Therefore, 3 nmol negative charge from plasmid DNA or RNA are associated with approximately 21 nmol positive charge from the [K]16-peptide. Lipofectin, however, provides only a further 0.25 nmol of positive charge. This suggests that, contrary to expectations, the enhancing effect of lipofectin in LID complexes is not charge related but may relate to the membrane destabilising effect of the DOPE component.
While not limited to the following theory of the mechanism of action, the following model of the early stages of the transfection process, which is based on the observations described herein, is proposed to explain the surprising and unexpected high efficiency of transfection by LID complexes, which high efficiency is found in all the cell types investigated.
The complexes are formed electrostatically by random association of lipofectin, oligolysine-peptide and plasmid DNA or RNA. The relative high proportion of oligolysine-peptide ensures a high proportion of integrin-targeting ligands per plasmid molecule. Particles are formed that contain one or more plasmids, associated with thousands of oligolysine-peptides and, therefore, a very high concentration of integrin-targeting ligands. By mixing lipofectin with the oligolysine-peptide, then adding plasmid DNA or RNA complexes are formed containing all three components. The particles, due to the high density of ligands, have a high avidity for integrins on cell surfaces, bind and are internalised by a phagocytic process (Hart et al., 1994). The vesicles fuse to form endosomes where, under acid conditions, the DOPE element contained within the particles mediates destabilisation of the endosomal membrane and subsequent plasmid release into the cytoplasm. Phagocytosed particles lacking lipofectin are degraded in the endosomes. Particles lacking the integrin-targeting moiety are less efficient at cell binding and internalisation. Both lipofectin and the oligolysine ([K]16) element of the oligolysine-peptides probably contribute to the overall efficiency of the LID complexes but the integrin-targeting capacity of the oligolysine/peptide component appears to be important for optimal targeting and internalisation of the complexes.